Conclusions

1
A new methodology to obtain wine yeast strains
overproducing mannoproteins
Quirós, M.1, Gonzalez-Ramos, D.2, Tabera, L.2,
Morales, P. 1, Pérez-Través, L.3, Barcenilla,
J.M.2 and Gonzalez. R.1
1 Instituto de Ciencias de la Vid y del Vino
(CSIC-UR-CAR), Logroño, Spain. 2 Instituto de
Fermentaciones Industriales (CSIC), Madrid,
Spain. 3 Departamento de Biotecnología. Instituto
de Agroquímica y Tecnología de los Alimentos
(CSIC), Burjassot, Spain.
Introduction Yeast mannoproteins are highly
glycosylated proteins that are covalently bound
to the ß-1,3-glucan present in the yeast cell
wall. These complex macromolecules are
structurally composed of polymers of sugars,
mainly mannose, that are covalently linked to
peptides and represent around 40 of the cell
wall dry weight. Yeast mannoproteins contribute
to several aspects of wine quality by protecting
against protein haze, reducing astringency,
retaining aroma and other undesired compounds
such as OTA and stimulating the growth of
lactic-acid bacteria. Therefore, the development
of a non-recombinant method to obtain enological
yeast strains overproducing mannoproteins would
be very useful. Previous works developed in our
group have allowed the identification of several
genetic determinants for the release of wine
yeast mannoproteins (Gonzalez-Ramos and Gonzalez,
2006 Gonzalez-Ramos et al., 2008). Among the
different recombinant genotypes constructed and
studied, strains deleted for KNR4 (from Killer
Nine Resistant, also known as SMI1), a gene
involved in ß-1,3-glucan biosynthesis yielded
optimal results (Gonzalez-Ramos et al., 2008). In
the present paper, a new methodology for the
selection of non-recombinant mannoprotein
overproducing yeast strains based on their
resistance to the killer 9 toxin of Williopsis
saturnus is developed.
Materials and methods UV Mutagenesis One haploid
laboratory strainis BY4741 and three industrial
winemaking strains IFI87, IFI475 and T73 were
used in this study. Overnight cultures were
washed twice and suspensions containing 107
cells/mL were then prepared, placed in a Petri
dish and irradiated under a UV lamp at 254 nm. 1
mL aliquots were taken after 20, 40 and 60 s of
exposure and kept in darkness for 30 min. Serial
10-fold dilutions of each of these aliquots (up
to 10-5) were prepared in saline solution and 100
µL were plated in YPD agar plates and k9
toxin-containing YPD plates. These were incubated
for 4872 h at 28 C and survival and resistance
rates for each of the exposure times were then
determined. k9 toxin-resistant mutants were
picked and transferred to fresh YPD plates. Ten
colonies were randomly picked both from the
control YPD plates and the selective YPD plates
containing k9 toxin in order to compare
polysaccharide and mannoprotein release with the
original strain after fermentation in GCY medium
The percentage of overproducing mutants obtained
for the industrial strains was not as stunning as
the observed for the haploid strain. However,
mutants producing promising results in GCY broth
were tested in synthetic must. Their fermentation
performance and the release of total
polysaccharides and mannoproteins compared to
their wild type counterparts are depicted in
Figs. 2A and B respectively.
Fermentation of GCY and synthetic must In order
to measure the improvement in the release of
mannoproteins, selected resistant strains were
inoculated into 100 mL shakeflasks containing 50
mL of GCY broth to a final OD600nm of 0.1 and
grown for 24 h at 30 C and 150 rpm. Cultures
were centrifuged and the supernatant transferred
to clean Falcon tubes. Macromolecules in these
supernatants were separated from monosaccharides
by gel filtration using Econo-Pac columns
(Bio-Rad). Industrial strains showing an
improved release of mannoproteins in GCY medium
were tested in a synthetic must (pH 3.5)
containing 10 glucose, 10 fructose, 0.6 citric
acid, 0.6malic acid, 0.17 YNB without
aminoacids and ammonium sulphate (Difco), 306
mg/L NH4Cl and 2/3 of the amount of aminoacids
used in a control synthetic grape must (CNC) by
Beltran et al. (2004). Fermentations were
performed in duplicate at 20 C using 100 mL
bottles capped with Müller valves containing 40
mL of must. Fermentation time course was
monitored by determining the production of CO2
expressed as weight loss until weight was
constant.
B)
Fig. 2. A) Time course of fermentations of
industrial wild type and mutant strains in
synthetic must. B) Total polysaccharides (mg/L)
released in synthetic must by the industrial
mutant strains selected in this work and their
wild type counterparts.
Once tested the fermentation performance and the
improved release of mannoproteins, we decided to
study their resistance to different stresses. As
seen in Fig. 3, none of the selected strains
presented a significantly sensitive phenotype to
any of the different stresses tested with regards
to their wild type counterparts.
Quantification of total mannoproteins and
polysaccharides The concentration of total
polysaccharides in the macromolecular fraction of
the supernantants was determined by the
phenol-sulfuric acid method described by Segarra
et al. (1995) using a standard curve of
commercial mannan from S. cerevisiae and the
concentration of mannoproteins determined by a
acid hydrolysis-HPLC method recently developed in
our lab (Quirós et al., In Press).
Results The normalized release of total
polysaccharides and mannoproteins in all the
k9-resistant strains tested is shown in Fig. 1 in
mg/(LOD). Eight out of the ten mutants
significantly produced more polysaccharides than
the control strain (plt0.01). The increase in the
production of polysaccharides and mannoproteins
ranged from 30 for mutant Mut3 to 238 for Mut6.
However, none of the colonies isolated from the
non-selective medium after the UV treatment
overproduced mannoproteins and total
polysaccharides (plt0.01, data not shown)
Conclusions The proposed methodology represents
a fast and straightforward approach to obtain
non-recombinant yeast strains overproducing
mannoproteins. The overproducing mutants present
similar fermentative performance to those of
their wild type counterparts and do not present a
hyper-sensitive phenotype to any of the stress
factors tested. An increased number of
interesting overproducing mutants could also be
obtained applying the described methodology to
spores isolated from the desired industrial
strains.
Fig. 1. Normalized production of polysaccharides
and mannoproteins (mg/LOD) obtained for ten
BY4741 mutant strains after UV mutagenesis,
selection on YPDk9 plates and fermentation in
GCY medium. Strains releasing a significantly
higher amount of mannoproteins than the wild type
strain are marked with an asterisk (plt0.01)

• References
• Gonzalez-Ramos and Gonzalez (2006) J. Agric.
  Food Chem. 54, 9411-9416.
• Gonzalez-Ramos et al. (2008) Appl. Environ.
  Microbiol. 74, 5533-5540.
• Beltrán et al. (2004) FEMS Yeast Res. 4,
  625-632.
• Quirós et al. Food Chem. doi10.1016/j.foodchem.2
  010.08.066